Project Summary As individual pathogens, tuberculosis (TB) and HIV infection are the leading infectious causes of death worldwide. The 1.5 million deaths from TB in 2014 account for only 5-10% of people infected with M. tuberculosis (Mtb), the bacteria that causes TB. This underscores the high prevalence of this infection that occurs by inhalation of the bacteria via aerosolized droplets. Co-infection with Mtb and HIV represent a ?deadly synergy?: During HIV infection, the peak of the viral set point and degree of viral heterogeneity is associated with poor outcome and patients with HIV-Mtb co-infection have been well recognized to have greater viral set points, diversity and faster progression to AIDS. Similarly, patients with Mtb-HIV co-infection have a higher risk of TB disease, reactivation TB and reinfection compared to those with Mtb alone. In our preliminary data, we show that the immune profiles of the airway cells are associated with the initial response to infection and outcome in an animal model that mimics human TB. These responses however, have not been fully characterized. We hypothesize that the early mucosal immune responses in the airway play important roles in the control of Mtb infection and this response is impaired during SIV (an HIV surrogate) infection. This proposal will leverage samples from already funded studies using an animal model that mimics both human Mtb and HIV infection (using SIV as a surrogate). In these studies, serial samples obtained before and during Mtb infection as well as subsequent SIV infection will be used. In Aim 1, we will identify signatures of airway protection against severe forms of TB during Mtb infection and SIV-Mtb co-infection. This will be done with a uniquely designed set of tools that profile innate and early adaptive mRNA transcriptional profiles. We will also better characterize innate lymphoid cells in the airways during the course of Mtb and SIV-Mtb co-infection. Data are emerging that viral divergence and evolution can occur in localized sites of co-infection. We hypothesize that localized sites of Mtb involvement (e.g., granulomas) in the lung and mediastinal lymph nodes can facilitate viral reservoirs where SIV viral growth and divergence can occur that ultimately leads to viral progression. In Aim 2, we will characterize the distribution of SIV viral diversity within and among Mtb specific lesions as well as peripheral sites of SIV involvement using MiSeq next generation and ultra- deep single genome sequencing. Results from these studies will reveal new innate and adaptive mechanisms of protection against severe forms of Mtb infection and how these are altered during SIV-Mtb co-infection that could ultimately be harnessed into new vaccine strategies. We will also characterized the depth of viral diversity within the SIV-Mtb co-infected host that could ultimately lead to better treatment strategies to limit the morbidity and mortality of these two pathogens.